Project Summary/Abstract Cancer cells have significantly altered glycosylation patterns compared to normal cells, but the functional significance with regard to cancer progression is not well understood. This gap in knowledge is largely attributable to the difficulties associated with studying cell-surface glycans and their associated structures. The need for new techniques to study glycoproteins is crucial to understand how aberrant glycosylation contributes to the onset, progression, and metastasis of cancer. Thus, the goal of this project is to develop enzymatic methods that allow for the study of cell-surface glycoproteins. E. coli secretes a mellaloprotease, secreted protease of C1-esterase inhibitor (StcE) that proteolytically cleaves mucin proteins on the surface of gut epithelial cells. Initial characterization of this enzyme confirmed previous reports of its ability to cleave mucin-type glycoproteins and its dependency on glycosylation. Further, StcE?s cleavage motif is dependent on both peptide sequence and the presence of glycosylation. As its motif is found within the vast majority of mucin-like glycoproteins, StcE and other bacterial mucinases can be used to open new avenues of basic research while also enabling therapeutic applications. This information will be used to interrogate aberrant glycosylation in two ways. First, natural killer cells express inhibitory receptors, sialic acid-binding immunoglobulin-like lectins (Siglecs), that downregulate NK cytotoxicity upon binding their sialic acid containing ligands. However, despite knowledge of glycan specificity, high-affinity glycoprotein ligands have remained elusive. StcE cleaves Siglec-7 glycoprotein ligands on the surface of tumor cells, as demonstrated by preliminary experiments. Thus, cell surface proteins will be treated with StcE. Cleaved peptides will be enriched with Siglec-7 conjugated beads and analyzed by mass spectrometry. Confirmed glycoprotein ligands will be analyzed for their ability to modulate NK activity. Secondly, mucin proteins are prevalent glycoproteins on the cell surface and are characterized by dense O- glycosylation that is abnormally truncated in cancer. However, it is not understood how tumors modulate structures and site-specificity of O-glycosylation. Using bacterial mucinases like StcE, these issues can be overcome. Four purported bacterial mucinases will be expressed and characterized. MUC1 and MUC16 will be isolated from overexpressing cell lines, bacterial mucinases will be used to digest the proteins in a site- and glycan-specific manner, and peptides will be analyzed with mass spectrometry. Additionally, the role of mucin degradation on immune function will be investigated. Ultimately, should the goals of this proposal be attained, the methods developed will prove invaluable to the field of glycobiology. Additionally, the biological information gathered will shed light on how glycosylation contributes to tumor progression, which will help in cancer immunotherapy design.